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From oncogenic replication stress to drug resistance : F-box proteins as signalling hubs in cancer

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posted on 2024-09-03, 00:07 authored by Andrä BrunnerAndrä Brunner

Cancer arises from cells that acquire genetic and epigenetic changes during the course of a, sometimes decades-long, somatic evolutionary process. These changes result in deregulation of a multitude of cellular processes leading to novel capabilities, often referred to as hallmarks of cancer, and a strong selective advantage for these cells albeit at a dramatic cost to the organism as a whole. Both, gene expression but also turn-over of gene products can become deregulated. The ubiquitin-proteasome system is responsible for the targeted degradation of proteins, and components of this system are altered during cancer development. Target specificity of this system is largely attained through E3 ubiquitin ligases that mediate the covalent attachment of ubiquitin to their substrates. The largest group of E3s are cullin-RING domain ligases (CRLs) with SKP1-cullin1-F-box protein (SCF) E3 ligases, or CRL1, representing one of the best-characterised subgroups of CRLs. These SCF ligases are multiprotein complexes containing one of, in human cells, 69 F-box proteins which function as substrate-adaptor subunits. Collectively, the family of F-box proteins has been found to be critically involved in virtually all the cancer hallmarks. However, despite their important role in cancer development, only a handful of SCFs has been molecularly and functionally well- characterised and detailed knowledge of how deregulation of specific SCF ligases and downstream substrate effectors impinges on cancer traits is lacking.

One of the main aims of the work presented in this thesis is to find cellular vulnerabilities resulting from deregulation of F-box proteins in cancer. FBXW7 is the most commonly mutated F-box protein in human cancers. Its inactivation leads to upregulation of its substrates including cyclin E, MYC or SOX9 (paper IV) resulting in deregulated proliferation, increased metastasis and drug resistance but also replication stress. Cancer cells undergoing replication stress become more dependent on signalling pathways detecting and repairing damaged DNA (papers I and III) and are consequently more sensitive to therapies targeting checkpoint and repair proteins such as WEE1, ATR or DNA-PK kinases (paper II).

In paper I we describe a novel function for the largely uncharacterised F-box protein FBXL12 in regulating the response to oncogene-induced replication stress. FBXL12 complements the Fanconi anaemia (FA) DNA repair pathway by targeting its central component FANCD2 for proteasomal degradation. The FA pathway not only plays a crucial role in resilience to endogenous sources of replication stress but also to drug-induced stress. FBXL12 and cyclin E are upregulated and correlated in human cancers and depletion of FBXL12 results in increased sensitivity to replication stress which posits FBXL12 as a potential cancer drug target. Ablation or pharmacological inhibition of FBXL12 prevents degradation of FANCD2 and breast cancer cells are sensitised to the adverse effects of drug- as well as oncogenic cyclin E-induced replication stress.

In paper II we focus on exploring further ways of sensitising cancer cells to replication stress. We performed a screen to identify potential viability markers in response to replication stress induced by WEE1 inhibitor AZD1775 and discover novel synergistic combinations. Additionally, we determine a subset of basal-like breast cancer cells that responds to treatment initially but recovers after treatment cessation and identify PTEN as a novel predictive marker for such responses, with cells expressing low levels of PTEN being highly sensitive acutely and failing to recover. Furthermore, inactivation or genomic deletion of DNA-PK, an apical DNA damage kinase, attenuates recovery and sensitises basal-like breast cancer cells to AZD1775. Mechanistically, loss of PTEN or DNA-PK impair CHK1 activation and S-phase arrest in response to AZD1775 treatment, which finally ensues lethal replication stress and loss of survival.

In paper III we concentrate on FBXO28, another poorly-studied member of the F-box family, which we find to degrade ARHGEF6 and ARHGEF7 activators of the Rho-type GTPase RAC1, involved in cell motility. Surprisingly, we identify a novel function for FBXO28 and ARHGEF6/7 in promoting the repair of breaks in heterochromatin DNA. Following DNA damage, tightly chromatin-bound FBXO28 is released and promotes degradation of nucleoplasmic ARHGEF6/7 to modulate activation and inactivation cycles of nuclear RAC1 and allow for efficient resolution of H3K9me2/3-positive damaged sites.

In paper IV we add a key oncogenic transcription factor to the growing list of FBXW7 substrates; SOX9. FBXW7 ubiquitylates and degrades SOX9 upon phosphorylation by GSK3β. Mutation and inactivation of FBXW7 in medulloblastoma concurs with elevated SOX9 protein expression and poor patient outcome. In medulloblastoma cell line models we demonstrate increased cell motility, metastasis and increased resistance to cytostatic treatment after expression of a non-degradable SOX9 mutant. Conversely, inhibition of the PI3K/AKT/mTOR pathway promoted GSK3β-dependent SOX9 degradation and sensitised FBXW7-proficient medulloblastoma cells to cisplatin.

List of scientific papers

I. Brunner, A.§, Johansson, H., Kourtesakis, A., Viiliäinen, J., Widschwendtner, M., Wohlschlegel, J., Lehtiö, J., Spruck, C., Orre, L.M., Rantala, J. K. and Sangfelt, O.§. Degradation of FANCD2 by SCF-FBXL12 alleviates cyclin E-driven replication stress and maintains genomic integrity. §Corresponding author. [Manuscript]

II. Brunner, A.*, Suryo Rahmanto, A.*, Johansson, H.±, Franco, M.±, Viiliäinen, J., Gazi, M., Frings, O., Fredlund, E., Lehtiö, J., Rantala, J. K., Larsson, L.-G. and Sangfelt, O§. PTEN and DNA-PK as determinants of sensitivity and recovery in response to WEE1 inhibitor AZD1775 in human breast cancer. eLife. 2020;9:e57894. *Equal contribution, ±Equal contribution, §Corresponding author.
https://doi.org/10.7554/eLife.57894

III. Čermák, L.*§, Brunner, A.*, Baloghová, N., Ueberheide, B., Ng, H.-F., Wohlschlegel, J., Manser, E., Sangfelt, O.±§ and Pagano, M±§. FBXO28 controls nuclear RAC1 activity and safeguards efficient heterochromatin DNA repair by targeting ARHGEF6/7 for degradation. *Equal contribution, ±Equal contribution, §Corresponding author. [Manuscript]

IV. Suryo Rahmanto, A.*, Savov, V.*, Brunner, A.±, Sara Bolin, S.±, Weishaupt, H.±, Malyukova, A., Rosén, G., Čančer, M., Hutter, S., Sundström, A., Kawauchi, D., Jones, D. T. W., Spruck, C., Taylor, M. D., Cho, Y.-J., Pfister, S.M., Kool, M., Korshunov, A., Swartling, F. J.§ and Sangfelt, O§. FBW7 suppression leads to SOX9 stabilization and increased malignancy in medulloblastoma. EMBO J. 35(20): 2192–2212. *Equal contribution, ±Equal contribution, §Corresponding author.
https://doi.org/10.15252/embj.201693889

History

Defence date

2021-02-19

Department

  • Department of Cell and Molecular Biology

Publisher/Institution

Karolinska Institutet

Main supervisor

Sangfelt, Olle

Co-supervisors

Swartling, Fredrik

Publication year

2021

Thesis type

  • Doctoral thesis

ISBN

978-91-8016-063-6

Number of supporting papers

4

Language

  • eng

Original publication date

2021-01-28

Author name in thesis

Brunner, Andrä

Original department name

Department of Cell and Molecular Biology

Place of publication

Stockholm

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